The DDB1-CUL4-RBX1 (CRL4) ubiquitin ligase family regulates a diverse set of cellular pathways through dedicated substrate receptors (DCAFs). The DCAF DDB2 detects UV-induced pyrimidine dimers in the genome and facilitates nucleotide excision repair. We provide the molecular basis for DDB2 receptor-mediated cyclobutane pyrimidine dimer recognition in chromatin. The structures of the fully assembled DDB1-DDB2-CUL4A/B-RBX1 (CRL4(DDB2)) ligases reveal that the mobility of the ligase arm creates a defined ubiquitination zone around the damage, which precludes direct ligase activation by DNA lesions. Instead, the COP9 signalosome (CSN) mediates the CRL4(DDB2) inhibition in a CSN5 independent, nonenzymatic, fashion. In turn, CSN inhibition is relieved upon DNA damage binding to the DDB2 module within CSN-CRL4(DDB2). The Cockayne syndrome A DCAF complex crystal structure shows that CRL4(DCAF(WD40)) ligases share common architectural features. Our data support a general mechanism of ligase activation, which is induced by CSN displacement from CRL4(DCAF) on substrate binding to the DCAF.

MSC have self-renewal and multilineage differentiation potential, including differentiation into endothelial cells and vascular smooth muscle cells. Although bone marrow-derived mononuclear cells (MNC) have been applied for therapeutic angiogenesis in ischemic tissue, little information is available regarding comparison of the molecular foundation between MNC and their MSC subpopulation, as well as their response to ischemic conditions. Thus, we investigated the gene expression profiles between MSC and MNC of rat bone marrow under normoxia and hypoxia using a microarray containing 31,099 genes. In normoxia, 2,232 (7.2%) and 2,193 genes (7.1%) were preferentially expressed more than threefold in MSC and MNC, respectively, and MSC expressed a number of genes involved in development, morphogenesis, cell adhesion, and proliferation, whereas various genes highly expressed in MNC were involved in inflammatory response and chemotaxis. Under hypoxia, 135 (0.44%) and 49 (0.16%) genes were upregulated (>threefold) in MSC and MNC, respectively, and a large number of those upregulated genes were involved in glycolysis and metabolism. Focusing on genes encoding secretory proteins, the upregulated genes in MSC under hypoxia included several molecules involved in cell proliferation and survival, such as vascular endothelial growth factor-D, placenta growth factor, pre-B-cell colony-enhancing factor 1, heparin-binding epidermal growth factor-like growth factor, and matrix metalloproteinase-9, whereas the upregulated genes in MNC under hypoxia included proinflammatory cytokines such as chemokine (C-X-C motif) ligand 2 and interleukin-1␣. Our results may provide information on the differential molecular mechanisms regulating the properties of MSC and MNC under ischemic conditions.

Acute autonomic and sensory neuropathy is a rare disorder that has been only anecdotally reported. We characterized the clinical, electrophysiological, pathological and prognostic features of 21 patients with acute autonomic and sensory neuropathy. An antecedent event, mostly an upper respiratory tract or gastrointestinal tract infection, was reported in two-thirds of patients. Profound autonomic failure with various degrees of sensory impairment characterized the neuropathic features in all patients. The initial symptoms were those related to autonomic disturbance or superficial sensory impairment in all patients, while deep sensory impairment accompanied by sensory ataxia subsequently appeared in 12 patients. The severity of sensory ataxia tended to become worse as the duration from the onset to the peak phase of neuropathy became longer (P<0.001). The distribution of sensory manifestations included the proximal regions of the limbs, face, scalp and trunk in most patients. It tended to be asymmetrical and segmental, rather than presenting as a symmetric polyneuropathy. Pain of the involved region was a common and serious symptom. In addition to autonomic and sensory symptoms, coughing episodes, psychiatric symptoms, sleep apnoea and aspiration, pneumonia made it difficult to manage the clinical condition. Nerve conduction studies revealed the reduction of sensory nerve action potentials in patients with sensory ataxia, while it was relatively preserved in patients without sensory ataxia. Magnetic resonance imaging of the spinal cord revealed a high-intensity area in the posterior column on T(2)*-weighted gradient echo image in patients with sensory ataxia but not in those without it. Sural nerve biopsy revealed small-fibre predominant axonal loss without evidence of nerve regeneration. In an autopsy case with impairment of both superficial and deep sensations, we observed severe neuronal cell loss in the thoracic sympathetic and dorsal root ganglia, and Auerbach's plexus with well preserved anterior hone cells. Myelinated fibres in the anterior spinal root were preserved, while those in the posterior spinal root and the posterior column of the spinal cord were depleted. Although recovery of sensory impairment was poor, autonomic dysfunction was ameliorated to some degree within several months in most patients. In conclusion, an immune-mediated mechanism may be associated with acute autonomic and sensory neuropathy. Small neuronal cells in the autonomic and sensory ganglia may be affected in the initial phase, and subsequently, large neuronal cells in the sensory ganglia are damaged.

SummaryRAD52 mediates homologous recombination by annealing cDNA strands. However, the detailed mechanism of DNA annealing promoted by RAD52 has remained elusive. Here we report two crystal structures of human RAD52 single-stranded DNA (ssDNA) complexes that probably represent key reaction intermediates of RAD52-mediated DNA annealing. The first structure revealed a “wrapped” conformation of ssDNA around the homo-oligomeric RAD52 ring, in which the edges of the bases involved in base pairing are exposed to the solvent. The ssDNA conformation is close to B-form and appears capable of engaging in Watson-Crick base pairing with the cDNA strand. The second structure revealed a “trapped” conformation of ssDNA between two RAD52 rings. This conformation is stabilized by a different RAD52 DNA binding site, which promotes the accumulation of multiple RAD52 rings on ssDNA and the aggregation of ssDNA. These structures provide a structural framework for understanding the mechanism of RAD52-mediated DNA annealing.

The genetic mutation of X-linked recessive bulbospinal neuronopathy is amplification of a polymorphic tandem CAG repeat in the androgen receptor gene. We studied this CAG repeat in 26 Japanese patients from 21 families with X-linked recessive bulbospinal neuronopathy. The number of CAG repeats was significantly correlated with the age at onset of limb muscular weakness (r = -0.596, p < 0.001) and age-adjusted scored disability (r = 0.446, p < 0.03). The length of the CAG repeat therefore seems to be a determinant factor of clinical severity.

Although carbon ion therapy facilities are expensive, the biological effects of carbon ion beam treatment may be better against cancer (and cancer

No abstract

SummaryThe mitogen-activated protein kinase (MAPK) cascade, consisting of MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK), is the signaling system that relays various external signals, including mitogens and stresses in eukaryotes. MAPKK is activated by phosphorylation in the consensus motif, SXXXS/T, in animals, but the regulation mechanism for the plant MAPKK by phosphorylation, having the putative phosphorylation motif of S/TXXXXXS/T, is not yet fully clari®ed. Here we constructed a series of mutants of AtMEK1, an Arabidopsis MAPKK, having the sequence T218-X-S220-X-X-X-S224 that ®ts both of the plant-and animal-type motifs. We show that the two double-mutant proteins replacing Thr-218/Ser-224 and Ser-220/Ser-224 by Glu expressed in Escherichia coli show a constitutive activity to phosphorylate the Thr and Tyr residues of the kinase-negative mutant of an Arabidopsis MAPK, named ATMPK4, in vitro. The mutation analysis of AtMEK1 replacing Thr-218 and Ser-220 to Ala suggested that Thr-218 is autophosphorylated by the enzyme. The wild-type ATMPK4 was also phosphorylated by the active mutants of AtMEK1 and showed a high protein kinase activity toward myelin basic proteins. In contrast, ATMPK3, another Arabidopsis MAPK, was a poor substrate of this plant MAPKK, indicating that AtMEK1 has a substrate speci®city preferring ATMPK4 to ATMPK3, at least in vitro. Furthermore, AtMEK1 immunoprecipitated from Arabidopsis seedlings stimulated with wounding, cold, drought, and high salt showed an elevated protein kinase activity toward the kinase-negative ATMPK4, while the amounts of the AtMEK1 protein did not change signi®cantly. These data indicate that the AtMEK1 becomes an active form through phosphorylation and activates its downstream target ATMPK4 in stress response in Arabidopsis.